The present invention relates to novel styralyl alcohol isomer mixtures and novel sytralyl acetate isomer mixtures, fermentation processes for preparing same and organoleptic uses for same as aroma or food flavor ingredients.
In today""s market, it is frequently desirable to identify flavor components of food items as being xe2x80x9cnatural flavors.xe2x80x9d It is generally recognized in the industry that a flavor compound having been prepared by microbial processes can be designated as a natural product and therefore have an important place in the commercialization of products containing them. As a result, the industry has devoted considerable time and effort to develop methods for the production of flavoring components and, in particular, for the production of certain alcohol and acetate derivatives which can properly be called xe2x80x9cnatural.xe2x80x9d
Furthermore, in today""s market, a trend is developing whereby it is now determined to be desirable to identify fragrance ingredients of fragrance compositions as being xe2x80x9cnatural fragrance ingredients.xe2x80x9d It is generally recognized in the industry that a fragrance compound having been prepared by microbial processes can be designated as a natural product and therefore have an important place in the commercialization of perfumes and perfumed articles containing them. As a result, the industry has devoted considerable time and effort to develop methods for the production of fragrance components and in particular for the production of alcohols and esters which can be called xe2x80x9cnatural fragrance ingredients.xe2x80x9d
It is also well known in the flavor and fragrance industry that particular stereoisomers of specific ingredients are, in many instances, more advantageous than their corresponding opposite stereoisomers. Accordingly, a significant amount of research has been carried out covering the formation of such stereoisomers using, for example, microbial reduction.
Thus, Simon, et al, Angew. Chem. Int. Ed Engl. 24 (1985), pages 539-553 (title: xe2x80x9cChiral Compounds Synthesized by Biocatalytic Reductionsxe2x80x9d) discloses at section 3.2 on page 545 the hydrogenation of ketones to form chiral secondary alcohols using Clostridia such as Clostridium kluyveri and specifically sets forth the production of xcex1-phenylethyl alcohol stereoisomers having the structure: 
with an enantiomeric excess of 94%.
Adlercreutz, Biotechnology Letters, Volume 13, No. 4 at pages 229-234 (1991) (title: xe2x80x9cASYMMETRIC REDUCTION OF KETONES WITH ENZYMES FROM ACETIC ACID BACTERIAxe2x80x9d) shows production of xcex1-phenylethyl alcohol at page 233 using G. oxydans (an enantiomeric excess of 99%); A. aceti (an enantiomeric excess of 92%); G. oxydans (an enantiomeric excess of 75%); A. aceti (an enantiomeric excess of 86%); A. pasteurianus (an enantiomeric excess of 94%); and A. peroxydans (an enantiomeric excess of 66%).
Nakamura, et al, Tetrahedron: Asymmetry, Volume 7, No. 10 at pages 3021-3024, 1996, published by Elsevier Science Ltd. (title: xe2x80x9cAsymmetric Synthesis of (S)-Arylatkanols by Microbial Reductionxe2x80x9d) sets forth the production of the isomers having the structures: 
using acetophenone as a starting material in accordance with the reaction: 
using Geotrichum candidum IFO 4597, but only shows the production of a stereoisomer mixture having 28% enantiomeric excess.
Vicenzi, et al, Enzyme and Microbial Technology, 20: pages 494-499, 1997, published by Elsevier Science Inc. (title: xe2x80x9cLarge-scale stereoselective enzymatic ketone reduction with in situ product removal via polymeric adsorbent resinsxe2x80x9d) discloses the stereoselective enzymatic reduction of 3,4-methylene-dioxyphenyl acetone to the corresponding S-3,4-methylene-dioxyphenyl isopropanol utilizing Zygosaccharomyces rouxii. 
Sorrilha, et al, Organic and Medicinal Chemistry Letters, Volume 2, No. 2 at pages 191-196, 1992, published by Pergamon Press plc (tide: xe2x80x9cREDUCTION OF PHENYLKETONES BY IMMOBILIZED BAKER""S YEASTxe2x80x9d) discloses a process wherein baker""s yeast immobilized on chrysotile and montmorillonite causes stereoselective reduction of 1-phenyl-1,2-propanedione to the corresponding (1R,2S)-diol.
Nothing in the prior art, however, discloses the production of the stereoisomeric mixture of the compounds having the structures: 
having an xcex1D20=xe2x88x9238.6xc2x0 with an enantiomeric excess percent of 87.5 ([xcex5xcex5%=87.5]) and furthermore, nothing in the prior art discloses the production of the stereoisomeric mixture of esters having the structures: 
having (xcex1D20=+73.06xc2x0) with an enantiomeric excess percent of 79.2, [xcex5xcex5%=79.2] or xcex1D20=xe2x88x92102.7xc2x0 with an enantiomeric excess percent of 89.15, [xcex5xcex5%=89.15], which materials have been found by us to have unexpected, unobvious and advantageous properties insofar as their organoleptic (flavor and fragrance) utilities are concerned.
The above and other objects and features of the invention are obtained in accordance with the present invention by carrying out a process using (i) reductive reaction techniques to produce and recover certain naturally occurring alcohols and (ii) esterification reaction techniques to produce and recover certain naturally occurring esters, found to be useful for their organoleptic properties in augmenting or enhancing the aroma or taste of consumable materials such as foodstuffs, chewing gums, toothpastes, oral care products, chewing tobaccos, smoking tobaccos, perfume compositions, colognes and perfumed articles such as solid or liquid detergents, perfumed polymers, fabric softener compositions, fabric softener articles, cosmetic powders, hair preparations and the like.
The reductive reaction products are styralyl alcohol stereoisomer mixtures containing compounds having the structures: 
produced according to a fermentation reaction whereby acetophenone is reduced according to the reaction: 
using a culture of Kluyveromyces polysporus ATCC 22028.
The resulting products may be used as is for their organoleptic properties or may be further reacted by means of esterification with acetic acid according to the reaction: 
The aforementioned esterification reaction may take place using such esterification catalysts as citric acid or may take place by means of a fermentation reaction using an ester-forming enzyme, preferably Candida antarctica esterase expressed in Asperigilus orzae, for example NOVOZYM(copyright) 435, a triacylglycerol hydrolase (E.C. No. 3.1.1.3) acting as an effective carboxylesterase. NOVOZYM(copyright) is a trademark of the Novo Nordisk A/S Organization of Novo Alle, 2880 Bagsvaerd, Denmark.
When carrying out the reductive fermentation reaction, to wit: 
the resulting product has an optical rotation of xe2x88x9238.6xc2x0, [xcex1=xe2x88x9238.6xc2x0] and an enantiomeric excess percent of about 87.5, [xcex5xcex5%=87.5xc2x10.5].
When carrying out the esterification reaction, to wit: 
using a citric acid catalyst, the resultant stereoisomer mixture has an optical activity of xe2x88x92102.7xc2x0, [xcex1=xe2x88x92102.7xc2x0] with an enantiomeric excess percent of about 78.31, [xcex5xcex5%=78.31xc2x10.5].
When carrying out the esterification reaction, to wit: 
using the enzyme Candida antarctica esterase expressed in Asperigilus orzae, specifically NOVOZYM(copyright) 435, a triacylglycerol hydrolase (E.C. No. 3.1.1.3) acting as an effective carboxylesterase, a mixture of stereoisomers having an optical activity of +73.06xc2x0 is produced ([xcex1=+73.06xc2x0]) with an enantiomeric excess percent of about 81.1, [xcex5xcex5%=81.1xc2x10.5].
The mixture of optical isomers of the styralyl alcohol having the structures: 
produced as set forth above having an optical rotation of xe2x88x9238.6xc2x0, [xcex1=xe2x88x9238.6xc2x0] with an enantiomeric excess percent of about 87.5, [xcex5xcex5%=87.5xc2x10.5] has a mild hyacinth, gardenia aroma with strawberry nuances. It is, accordingly, useful in augmenting, enhancing and imparting aroma in or to floral fragrances and is useful in the creation of strawberry-flavored foodstuffs; e.g., strawberry-flavored gelatin desserts, strawberry-flavored chewing gums and strawberry-flavored fruit preparations for yogurt.
The mixture of stereoisomers of esters having the structures: 
prepared as set forth above using a citric acid catalyst and having an optical rotation of xe2x88x92102.7xc2x0, [xcex1=xe2x88x92102.7xc2x0] and an enantiomeric excess percent of about 78.31, [xcex5xcex5%=78.31xc2x10.5] has a fresh strawberry, green, dried fruit aroma with a green, avocado taste profile and strawberry jam nuances. From a fragrance standpoint, this substance is described as having a strawberry, green, dried fruit aroma with strawberry topnotes.
The mixture of stereoisomeric esters having the structures: 
prepared as set forth above using the ester-forming enzyme, Candida antarctica esterase expressed in Asperigilus orzae, and having an optical rotation of +73.06xc2x0, [xcex1=+73.06xc2x0] with an enantiomeric excess percent of about 81.1, [xcex5xcex5%=81.1xc2x10.5] has a fruity, floral, jasmine, mimosa, gardenia aroma with apricot, apple and strawberry jam flavor nuances. This material is also useful in the formation of fruity, floral, jasmine fragrances, as well as strawberry-flavored foodstuffs and food preparations such as strawberry-flavored gelatin desserts, strawberry-flavored ice cream and strawberry-flavored preparations for yogurts.
In carrying out the process for production of the styralyl alcohol stereoisomer mixtures containing stereoisomers having the structures: 
an inoculum preparation is first prepared containing a culture of Kluyveromyces polysporus ATCC 22028 and also containing nutrients including dextrose. The inoculum, after incubation, is then placed in a production fermenter which is aerated and agitated at, for example, an aeration rate of 0.25 v/v/m; at a temperature of, for example, 25xc2x0 C.; and at an agitation rate of, for example, 300 rpm for a time period of, for example, 24 hours. As set forth in the Detailed Description of the Invention, infra, the pH range may vary from about 5.5 up to about 6.
The resulting product is then extracted with a solvent such as ethyl acetate, and the solvent extract is then washed in order to bring the pH up to approximately neutral (pH=7). The resulting washed extract is then concentrated and the resulting concentrate is then fractionally distilled. The resulting distillate is then used xe2x80x9cas isxe2x80x9d for its organoleptic properties or further reacted with acetic acid according to the reaction: 
When carrying out the reaction using a citric acid catalyst, the resulting distillate is admixed with acetic acid and citric acid, and the resulting mixture is refluxed for about 15 hours while excess acetic acid is recovered. At the end of the 15 hour period, the resulting product is fractionally distilled.
When carrying out the esterification reaction, to wit: 
using the ester-forming enzyme, Candida antarctica esterase expressed in Asperigilus orzae, specifically, NOVOZYM(copyright) 435, the mixture of styralyl alcohol stereoisomers having the structures: 
is admixed with a small amount of water and a small amount of NOVOZYM(copyright) 435. The resulting mixture is heated to 40xc2x0 C. and acetic acid is then added over a period of between about 5 and about 10 hours and then continuously stirred for an additional 40-60 hours. The conversion using the xe2x80x9cenzymexe2x80x9d process is between about 2.5 and about 3.5%. The resulting product is then fractionally distilled at a pressure of 5 mm/Hg. and at a head temperature of 34-38xc2x0 C.